Various techniques have been developed for promoting in vitro mutagenesis on DNA sequences. Among these, mention may be made of site-directed mutagenesis and random mutagenesis.
Site-directed mutagenesis is a method which consists in altering the structure of a protein, in vitro, by simple modification of targeted codons in the sequence of the DNA. Thus, amino acids can be substituted at known or supposed active sites of the protein.
Random mutagenesis in vitro consists in introducing, during a replication or recombination step, mutations distributed randomly over the sequence of a gene or of a gene fragment. The mutations can be introduced throughout the length of the coding region of a gene, or can be confined to quite specific DNA segments. Unlike site-directed mutagenesis, a precise knowledge of the structure of the protein is not necessary to carry out random mutagenesis.
Several methods of random mutagenesis have been developed.
In a first, widely implemented approach, the polymerase chain reaction (PCR) is used under conditions which promote the introduction of mutations (error-prone PCR). With Taq polymerase, the frequency of base substitution can reach 10−3, i.e. 1 substitution per 1 000 base pairs (Moore et al. (1996) “Direct evolution of para-nitrobenzyl esterase for aqueous-organic solvant” Nat. Biotech. 14: 458-467). In other cases, a frequency of approximately 1 substitution per 500 bases has been reached (Sweasy et al. (1993) “Detection and characterization of mammalian DNA polymerase β mutants by functional complementation in Escherichia coli” Proc. Natl. Acad. Sci. USA 90: 4626-4630; Diaz et al. (1991) “PCR-mediated chemical mutagenesis of cloned duplex DNAs”. Biotechniques 11: 204-211).
However, this technique has several drawbacks. In particular, it requires a step in which the substrate to be modified is treated with genotoxic chemical compounds. In addition, it produces too low a rate of mutagenesis. It is not therefore suitable for the construction of a library of mutants and the rapid selection of advantageous mutants.
In another approach, use is made of oligonucleotides (for example 19 or 47 base pairs) of random sequence synthesized by a chemical process (degenerate oligonucleotides), which are inserted into a gene, preferably into the region encoding the active part of the enzyme, and used to provide a diversity of proteins. (Horowitz et al. (1986) “Promoters selected from random DNA sequences” Proc. Natl. Acad. Sci. USA 83: 7405-7409; Dube et al. (1989) “Mutants generated by the insertion of random oligonucleotides into the active site of the β-lactamase gene” Biochemistry 267: 5703-5707). This procedure requires the synthesis, also expensive, of many mutated oligonucleotides.
Yet another approach, based on the use of homologous recombination, similar to the natural process of genetic mixing which takes place during evolution, can be used. This method is called shuffling (Stemmer (1994) “DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution” Proc. Natl. Acad. Sci. USA 91: 10747-10751). It consists in carrying out a PCR on fragments of a gene or fragments of several homologous genes, subsequent to a random digestion with DNAse I. The small fragments derived from this digestion serve as primers with respect to one another during the PCR, and lead to the introduction of random mutations by recombination. This method is laborious and takes place in several steps (digestion of the DNA, recombination by PCR in the presence of “mega-primers”, etc.), which involves difficulties in implementation. It has been used in improving the activities of several enzymes such as Green Fluorescent Protein, β-lactamase, and also the arsenate detoxication operon.
The fidelity of several DNA polymerases has been tested in order to measure their mutagenic capacity. Among these, the Klenow fragment of Escherichia coli DNA polymerase I, T4 DNA polymerase, the T7 phage DNA polymerase sequenase, and the Taq DNA polymerase. Among all these polymerases, the Taq polymerase exhibits the least fidelity. However, the frequency of mutagenesis remains too low to envisage its use for random mutagenic purposes without modification of the reaction conditions or of the polymerase itself (Cadwell et al. (1992) “Randomization of genes by PCR mutagenesis” Cold Spring Harbor Laboratory 2: 28-33).
Consequently, despite the techniques developed, there remains, to date, a need for a random mutagenesis technique which is simple to implement, which does not involve the use of genotoxic chemical compounds or of complex steps, or the synthesis of numerous oligonucleotides, and which generates a sufficiently high frequency of random mutations to envisage the creation of a library of proteins.